The Root Extract of the Medicinal Plant Pelargonium sidoides Is a Potent HIV-1 Attachment Inhibitor

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs.     

Wind Farm Facilities in Germany Kill Noctule Bats from Near and Far

Over recent years, it became widely accepted that alternative, renewable energy may come at some risk for wildlife, for example, when wind turbines cause large numbers of bat fatalities. To better assess likely populations effects of wind turbine related wildlife fatalities, we studied the geographical origin of the most common bat species found dead below German wind turbines, the noctule bat (Nyctalus noctula). We measured stable isotope ratios of non-exchangeable hydrogen in fur keratin to separate migrants from local individuals, used a linear mixed-effects model to identify temporal, spatial and biological factors explaining the variance in measured stable isotope ratios and determined the geographical breeding provenance of killed migrants using isoscape origin models. We found that 72% of noctule bat casualties (n = 136) were of local origin, while 28% were long-distance migrants. These findings highlight that bat fatalities at German wind turbines may affect both local and distant populations. Our results indicated a sex and age-specific vulnerability of bats towards lethal accidents at turbines, i.e. a relatively high proportion of killed females were recorded among migratory individuals, whereas more juveniles than adults were recorded among killed bats of local origin. Migratory noctule bats were found to originate from distant populations in the Northeastern parts of Europe. The large catchment areas of German wind turbines and high vulnerability of female and juvenile noctule bats call for immediate action to reduce the negative cross-boundary effects of bat fatalities at wind turbines on local and distant populations. Further, our study highlights the importance of implementing effective mitigation measures and developing species and scale-specific conservation approaches on both national and international levels to protect source populations of bats. The efficacy of local compensatory measures appears doubtful, at least for migrant noctule bats, considering the large geographical catchment areas of German wind turbines for this species.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments.     

Expression and Localization of Lung Surfactant Proteins in Human Testis

Background

Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions.

Objective

The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated.

Results

Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig.

Conclusion

Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP''s was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.     

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.     

Aggregation of misfolded proteins can be a selective process dependent upon peptide composition

Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.     

Support for association of schizophrenia with genetic variation in the 6p22.3 gene,dysbindin, in sib-pair families with linkage and in an additional sample of triad families

Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach.     

Cholesterol: free radical peroxidation and transfer into phospholipid membranes

Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.